Bacillaceae Spores, fungi and aflatoxins Determination in Honey

Pendahuluan

Honey is Komposisi utama madu adalahmainly composed by sugars, particularly the mono- gula, khususnya monosaccharides fructose and glucose, though it containssakarida,fruktosa dan glukosa, walaupun berisi sebagiana large variety of di-and trisaccharides (White, 1983). besar dari berbagai  trisakarida (White, 1983).

Microbiological characteristics of honey are inherent to quality andCiri-ciri mikrobiologi dari madu sangat melekat pada kualitas dansafety. keselamatan. The microbes of concern in honey are prima- Mikroba yang cukup diperhatikan pada madu yang penting adalah yeast, jamur dan bakteri pembentuk spora. TheseMikroorganisme ini mungkin terlibat dalam proses produksi enzim, antibiotics, mycotoxins and growth factors (vitamins,biotics, mycotoxins dan faktor pertumbuhan (vitamin,amino acids), metabolic conversion of provisions, and asam amino), metabolisme konversi dari ketentuan, daninhibition of competing microorganisms (Goerzen, inhibisi dari persaingan seperti mikroorganisme,dan dapat menyebabkanspoilage of provisions, production of enzymes, anti- kerugian(Goerzen,1991). 1991). Honey has been incriminated as a source of C. Madu telah dididentifikasi sebagai sumber C.botulinum spores responsable for infant botulism cases spora botulinum yang menginap pada bayi dalam kasus infant botulism (Amon et al., 1981).(Amon dkk., 1981). A different clinical form of botu-Secara klinis berbagai bentuk botulism, has been recently recognised in infants under 1lism, baru-baru ini telah ditemukan pada bayi di bawah 1year of age. tahun keatas. In this type of botulism spores of bacteria Dalam jenis ini botulism spora dari bakteriapparently germinate and produce toxin in the intesti- tampaknya mulai tumbuh dan menghasilkan toksin dalam intestinal tract of affected infants (Huttanen et al., 1981).nal yang berpengaruh pada bayi (Huttanen dkk., 1981).Pollen may be the original source of microbes for

Persentase kontaminasiBacillus cereus , and with fungi: yeasts, Mucor sp, Bacillus cereus, dan dengan cendawan: ragi, Mucor sp,Penicillium spp and several species of genus Asper- Penicillium spp dan beberapa spesies dari genus Aspergillus , particularly Asp.gillus, terutama Asp. flavus , Asp. flavus, Asp. candidus, Asp. candidus, Asp.fumigatus and Asp. fumigatus dan Asp. niger . niger. These potentially pathogenic Mikroba ini berpotensi secara patogenikspecies can be harmfull to predisposed patients. dan dapat membahayakan konsumen Di antaramycotoxins, aflatoxins could pose a significant threat mycotoxins, aflatoxins signifikan dapat menimbulkan ancamanto human health because they are toxigenic, carcinoge- kesehatan manusia karena bersifat toxigenic, carcinogenic, mutagenic and teratogenic (Hsieh, 1986), besidesnic, mutagenik dan teratogenic (Hsieh, 1986).

Deteksi C. perfringens spores perfringens spora

For the enumeration of Clostridium perfringens ,            Clostridium perfringens, 1 ml of each decimal dilution was incorporated onto1 ml dari setiap pencairan desimal telah dimasukkan ke Tryptose sulfite cycloserine Agar (TSC) (Oxoid code Tryptose sulfite cycloserine Agar (TSC) setelahthermal treatment at 80 ºC for 10 minutes (ISO, 1995). thermal perawatan di 80 º C selama 10 menit (ISO, 1995), di bawahanaerobic conditions, for 48 h at 45 ºC. kondisi anaerobik, untuk 48 h di 45 º C.

Mycological examination Mycological pemeriksaan

For enumeration and identification at genus levelIdentifikasi di tingkat genusof moulds and yeast, 1 ml of honey of each dilution dari jamur dan ragi, 1 ml madu setiap pencairanwas spread into each of five plates (0.2 ml/plate) of telah menyebar ke setiap lima piring (0,2 ml / plate) dariGlucose Yeast Extract Sucrose Agar (GYES) (King et Glukosa ragi Sucrosa Extract Agar (GYES) (Raja etal ., 1984) and incubated at 28 ºC for 3-5 days. al., 1984) dan incubated di 28 º C selama 3-5 hari. Each Tiap isolated mould colony was observed microscopically koloni mould telah diamati secara mikroskopisfor morphological characterization and identification berdasarkan karakterik morfologi dan identifikasi.

Determination of aflatoxins B1, B2, G1 Penentuan aflatoxins B1, B2, G1and G2 by HPLC dan G2 oleh HPLC

The solvent mixture was water +            Larutan adalah campuran air + methanol (8+2) instead of methanol+water (8+2,v/v).methanol (8 +2). Batas The detection limit was 1µg/kg.deteksi adalah sebesar 1μg/kg. The sample extract Sampel sariwas filtered, diluted and applied in an immunoaffinity telah disaring, diencerkan dan diterapkan dalam sebuah immunoaffinitycolumn containing antibodies specific to aflatoxins kolom yang berisi antibodi spesifik untuk aflatoxinsB1, B2, G1 and G2. B1, B2, G1 dan G2. Standard AFs B1, B2, G1 and Standar AFS B1, B2, G1 danG2 were purchased from Sigma-Aldrich (Ref. A-6636, G2 dibeli dari Sigma-Aldrich (Ref. A-6636,A-9887, A-0138 and A-0263 respectively) (Quimica A-9887, A-0138 dan A-0263 masing-masing) (QuimicaSA Spain). SA Spanyol). The stock solution, working standards andThe recoveries werePemulihan yang telahdone in duplicate, in blank samples of honey (1kg), dilakukan di duplikat, pada sampel madu murni (1kg), spiked with levels of 4.0 mg/kg of AFB1 and AFG1didapatkan pada level 4,0 mg / kg AFB1 dan AFG1,and 2.0 mg/kg of AFB2 and AFG2. dan 2.0 mg / kg AFB2 dan AFG2. The average reco- Rata-rata-emilgiglioveries were 89.5 % for AFB1, 87.0% for AFB2, 88.3 89,5% yang veries untuk AFB1, 87,0% untuk AFB2, 88,3 % for AFG1 and 86.2 % for AFG2. Untuk AFG1% dan 86,2% untuk AFG2. Samples with AFs Sampel dengan AFSlevels below 1 mg/kg were considered negative (infe- tingkat di bawah 1 mg / kg dianggap negatif.

Results Hasil

From the 80 samples, only nine do not revealed any ElevenMartins, HM et al.of the samples (13.8%) were contaminated with sporesDari sampel madu(13,8%) telah terkontaminasi oleh spora of B.dari spesies B. cereus , five (6.2%) of which had levels lower cereus, lima sampel (6,2%) dari tingkat yang lebih rendah than 101022 spora/gram, five samples (6.2%) with levels greater than, Lima sampel (6,2%) dengan tingkat lebih besar sebesar 101033 cfu/g; one sample (1.3%) revealed to be contami-spora/gram; satu sampel (1,3%) yang akan menyebabkan contaminasinated with 10 sebesar 1055 spores/g.spora /gram. Spores of C.

Spora dari C. perfringens were perfringens tidak terdeteksi pada setiap sampel (1 g). Of the 80 samples analysed, 71(88.8%) were conta-Dari 80 sampel analisis, 71 (88,8%) adalah tercontaminated with fungi; of these samples, 46 were conta-minasi dengan mould, 46 orang-terkontaminated with moulds and yeasts, and 25 samples pre-minasi oleh jamur dan ragi, dan 25 sampel terksented only yeasts.ontaminasi oleh  ragi. The moulds identified were: Asp. Jamur yang diidentifikasi adalah: Asp.candidus (28.7%), Asp. candidus (28,7%), Asp. flavus (57.5%), Asp. flavus (57.5%), Asp. fumigatus fumigatus(45.0%), Asp. (45,0%), Asp. niger (51.3%), Mucor sp. niger (51,3%), Mucor sp. (31.3%) and (31.3%) danPenicillium spp. Penicillium spp. (38.8%), with levels ranging from 10 (38,8%), Two yeast species were identified: Candida humi-Dua ragi spesies dikenali: Candida humicola (75.0%) and Saccharomyces sp.cola (75,0%) dan Saccharomyces sp. (88.8%); Its level (88,8%)

.

Discussion Kesimpulan

Huttanem et al. (1981), in 80 honey samples collec-            Huttanem dkk. (1981), dalam 80 sampel maduted from apiaries in Pennsylvania, Illinois and New dilaporkan tidak ditemukan Clostridia spora. In another Di lainstudy, Kautter et al. (1982), in 100 samples of honey studi, Kautter dkk. (1982), di 100 sampel madufound only two samples (2.0 %) contaminated with ditemukan hanya dua sampel (2,0%) tercemar denganClostridium botulinum spores. Clostridium botulinum spora. Dari hasil studiThe present study does not revealed contamination superior to 1 spore/g (limit tidak menunjukkan kontaminasi unggul pada 1 spora / g.Hauschild et al. (1988), state that honey generally Hauschild dkk. (1988), menyatakan bahwa madu umumnyacontains few botulinic spores. berisi beberapa botulinic spora. According to Bonvehi The results of the Hasilpresent study revealed contamination by B. hadir studi menunjukkan kontaminasi oleh B. cereus in cereus di11 samples; one of the samples had a heavy contami- 11 sampel; Potensi toxigenic efeknyaachieved on levels above 10 dicapai pada tingkat di atas 1055spores/g. spora / g.Jiménez et al ., (1994), studying raw honey referred mycoflora dominan termasuk Aspergillus flavus, Asp. flavus, Asp. niger, Asp. niger, Asp. candidus, Asp. candidus, Asp. terreus, Peni- terreus, Penicillium spp., Saccharomyces sp. and Zygosaccharomy- cillium spp., Saccharomyces sp. dan Zygosaccharomyces . ces. This study partially confirms this findings. Spesies yeast yang The yeast species identified ( Candida humicolaidentifikasi adalah Candida humicolaand Saccharomyces sp.) were detected in a very high dan Saccharomyces sp. yang terdeteksi dalam frequency and at high levels of contaminatfrekuensi tinggi dan di tingkat kontaminasi yang tinggi. OThis osmophylic yeast are probably good indicators forsmophylic yeast ini mungkin baik untuk indikatormicrobiological quality of honey. microbiological kualitas madu.There are few informations concerning mycological Ada beberapa informasi tentang mycologicalcontamination and simultaneous co-occurrence of Asp. kontaminasi yang bersamaan terdapat di dalamnya Aspflavus or Asp. flavus atau Asp. parasiticus and aflatoxins detection in parasiticus dan aflatoxinshoney.. Hilldrup et al. (1977), studied fungal growth Dapat disimpulkan bahwamicrobial contamination levels in honey is generally kontaminasi mikrobial di tingkat madu pada umumnyalow. rendah. It is not surprising to find that none of the stains of Asp. Asp. flavus have produced aflatoxins, because the pro- flavus telah menghasilkan aflatoxins, karena production depends not only of the genetic competence ofvaksin tidak hanya tergantung dari genetik kompetensi darithe strains, but is also influenced by a quite wide range strains, tetapi juga sangat dipengaruhi oleh berbagaiof factors (substrate composition, very low aW , and faktor antara lain komposisi substrat, AW yang rendah,acidity of honey) and ecological conditions. Tingkat keasaman madu dan kondisi ekologi.

Studi ini menyatakan adanya kontaminasi madu oleh Bacillaceaespores ( Clostridium perfringens, Bacillus cereus ), fungi and spora (Clostridium perfringens, Bacillus cereus), dan jamuraflatoxins. aflatoxins. The microflora was determined using conventional Microflora yang telah ditentukan menggunakan konvensionalmicrobiological methods and the aflatoxins were detected by microbiological metode dan aflatoxins yang terdeteksi oleh“high performance liquid chromatography” (HPLC). “Kromatografi cair berperforma tinggi (HPLC)”. Tetapi Spores Sporaof Clostridium perfringens were not detected in any sample. dari Clostridium perfringens tidak terdeteksi di setiap sampel.Bacillus cereus were identified in eleven samples (13.7 %); of Bacillus cereus ditentukan dalam sebelas sampel (13,7%); darithese, five (6.2%) had levels lower than 10 ini, lima sampel (6,2%) memiliki tingkat lebih rendah dari 1022 , five (6.2%) samples, Lima sampel  (6,2%) contained levels ranging from 10 terdapat mulai dari tingkat 1033 – to 101044cfu/g and only one spora/gram dan hanya satu sampel(1.3%) presented contamination above 10 (1,3%) terkontaminasi disajikan di atas 1055 cfu/g.spora/g. Yeasts and Ragi dan moulds were detected in 71 samples (88.8%).jamur yang terdeteksi pada 71 sampel (88,8%). Three genera of Tiga dari spesiesmoulds ( Aspergillus , Penicillium and Mucor ) and two genera of jamur (Aspergillus, Penicillium dan Mucor) dan dua dari spesies yeasts ( Saccharomyces and Candida ) were identified.yeast (Saccharomyces dan Candida) diidentifikasi. The most Persentase yang paling besar dari spesies prevalent among Aspergillus was A. Aspergillus adalah A. flavus (57.5%), followed flavus (57.5%), diikutiby A. oleh A. niger (51.3%), A. niger (51,3%), A. fumigatus (45.0%) and A. fumigatus (45,0%) dan A. candidus candidus(28.7%). Penicillium spp. (28,7%). Dari spesies Penicillium spp. and Mucor sp. dan Mucor sp. Persentase nya sebanyak were present in 38.8 38,8and 31.3 % respectively. Saccharomyces sp. dan 31,3%. Saccharomyces sp. and Candida humi- dan Candida humcola had high incidence (88.8 and 75.0 % respectively).cola memiliki tingkat insiden 88,8 dan 75,0%. None of Tidak satupunsamples revealed to be contaminated with aflatoxins. Sampel terkontaminasi oleh alflatoxins.

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